Integration of heterologous polynucleotides into the genomes of mammalian cells is routinely practiced for therapeutic purposes (e.g., gene therapy) and in the production of useful proteins or polypeptides in vitro. Insertion at random locations in the genome by non-homologous recombination requires several rounds of selection and clonal expansion to produce an acceptable expression system. The approach also needs to be repeated every time an expression system for a new gene is sought. Due to the random nature of the integration event, some of the locations where recombinant genes are inserted are incapable of supporting transcriptional events at all. This is because expression levels are greatly influenced by the effects of the local genetic environment at the gene locus (position effects). In addition, expression from many chromosomal sites is decreased over time. In some cases, this instability is due to DNA methylation of the transgene. As a result, wide variations in the expression level of integrated genes can occur, depending on the site of integration. In addition, random integration of exogenous DNA into the genome can in some instances disrupt important cellular genes, resulting in an altered phenotype.
Other than random insertion, recombinase-mediated integration has been described for insertion of transgenes at defined sites in the genome. However, achieving stable, high-efficient expressions of integrated transgenes is still cumbersome and requires large numbers of screened clones in order to select desirable integrated cells.
There is a need in the art for means for achieving a stable integration and/or high level of gene expression of heterologous polynucleotide in mammalian cells. The present disclosure addresses this and other needs.